In Vitro And Vivo Antibacterial Effect Of Borneol In Combination With Antibacterial Agents

Since penicillin was discovered,antibacterial drugs occupies anti-infection drugsmarket in clinical application gradually.It is like a double-edged sword, on the one hand,the escort for human health, has saved the life of tens of thousands of human beings;on theother hand,it had led to the spread of drug-resistant bacteria due to the unreasonable use ofantimicrobial agents,so a series of serious consequences come out,such as super bacteriaproduced and drug-induced diseases, etc.In a word,bacterial drug resistance has arousedwidespread attention in the field of global health.According to the survey,in the past70years,scientists have developed more than200kinds of antibacterial drugs,it means almostall of fungi that can produce antimicrobial active substances has been found, it led to thereduction of the types of antibiotics gradually in recent years.However, drug resistance ofbacteria to antibiotics is its inherent features, and the speed of the resistance is much fasterthan the speed of antibacterial drugs be discovered. The serious consequences of thespread of drug resistance will make us return to the age of no antibiotics, and it will lead tothe failure of the treatment of antibacterial drugs in anti-infection eventually. However,good curative effect and little side effects of antimicrobial drugs determined theirreplaceable effect in the field of anti-infective. Therefore, in order to keep applicationstrategy sustainably, strengthen the supervision of antimicrobial drugs application system,it is imperative to develop new types of antimicrobial agents to reverse bacterialresistance.In recent years,with the extensive and further study of traditional chinese medicineresources in the medical community, the effect of reverse bacterial drug resistance has been concernd gradually. Under the guidance of the systematic theory, there were a lot ofpractice showed that traditional Chinese medicine compound has some obviousadvantagesin antibacterial,delay or even eliminate bacteria resistance, this researchprovide the possibility to develop new antimicrobial agents.Borneol also called plum blossom borneol,can be divided into natural borneol andsynthetic borneol,the former can be extracted from the borneol plant Dryobalanopsaromatica Gaertnf. resin or compositae plant Blumea balsamifera DC. Leaf;the latter isalso called mechanism borneol,which can be chemical syntheticed by camphor orturpentine oil.Owing to the complicated composition and different sources,high purityd-borneol can be got from borneol fragrance plants,which contain a variety ofterpenoids,while einar incense mainly contains l-borneol,synthetic borneol is compose ofborneol and isoborneol and its mixture form raceme."Ben cao yan yi" recorded that borneol are "alone is weak, assist to make the active",which can develop humans,intelligence and wake up fast from sleeping in ancient ages.Alarge number of studies have shown that borneol can be two-way regulate centralnervous system, improved the bioavailability,resist the effect ofbacterial,inflammatory,virus and reversed the multi-drug resistance of tumor, etc. Itreported that borneol has an obvious antibacterial effect on staphylococcus aureus,escherichia coli,b-hemolytic streptococcus,etc.Low concentration of it can inhibit thegrowth of bacteria,while high concentrations can kill bacteria.In addition,borneol canpromote the absorption of gentamicin and increase its central and blood concentration, butthere were no report about joint antibacterial effect between antibacterial drugs andborneol to clinical common pathogenic bacteria,also no compound preparations appearedon the market.So in this subject, joint antibacterial effect between antibacterial drugs andborneol to clinical common pathogenic bacteria were discussed.In this article,the joint antibacterial effect in vivo and in vitro of borneol and clinicalcommonly used antimicrobial agents are studied，four kinds of Drug-resistant strains ofthe disease required，Staphylococus aureus，Escherichia coli,Pseudomonasaeruginosa，Klebsiella pneumoniae，respectively. All of them come from Guangdong hospital of dade road institute of clinical bacteria laboratory.(1)Bacteria counting. The concentration of bacteria suspension can be determined by photoelectric counting turbidimetric method,using UV spectrophotometry measure bacteria suspension absorbance,and the number of colony forming unit of bacteria suspension diluent can be studied by coated board counting method at the same time.On the basis of it, an A-cfu standard curve can be drawed,bacteria suspension can also be quanlited according to the absorbance of it in following studies.TheA-cfu standard curve of four kinds of test strains were as follows:Staphylococus a-ureus,y=(297.68x-56.990)×107,R2=0.9988;Escherichia coli,y=(528.15x-133.37)×106,R2=0.9978;Pseudomonas aeruginosa,y=(321.85x-45.411)×107,R2=0.9966;Klebsiella pneumo-niae,y=(257.63x-0.4971)×106,R2=0.9986,all standard curve had a good correlation.(2)Bacteria sensitivity measurement.With K-B paper method, the diameter of thebacteriostatic circle of clinical commonly used antimicrobial drug susceptibility to teststrains can be got,and then compared with the standards of American association ofclinical laboratory standardization (CLSI,2010edition).The results showed thatStaphylococus aureus(NO.13583) is a methicillin-resistant strain which showed obviousresistance to all kinds of beta lactam antibiotics and part of sulfa antibiotics;Escherichiacoli(NO.13920) was resist to all cephalosporin,most of penicillin class ofantibiotics,aztreonam and some sulfa antibiotics,so its resistance must be noticed;Pseudomonas aeruginosa(NO.13930)has an obvious resistance to some sulfa antibiotics,have high resistance to the third and fourth generation cephalosporin antibiotics；Klebsiella pneumoniaey(NO.13952) shown obvious resistance to most of antimicrobialagents,including all the class of penicillin,cephalosporin and aztreonam,this meant thatbacteria may produce extended spectrum beta-lactamases(ESBLs).(3)To Provide theoretical basis for the research on the body,research on the jointantibacterial effect of borneol and antibacterial agents in vitro,and screening combinationwhich had synergistic antibacterial effect and determine the the best ratio between themshould be carried out.First,using tube double dilution method to sduty the singleantibacterial effect of test strains of borneol and clinical commonly used antimicrobial drug and determined the minimum bacteriostatic concentration (MIC),respectively.Second,to determinate the joint MIC of them,trace checkerboard dilutionmethod is used to examine antimicrobial effect of borneol associated with antimicrobialagents.Finally calculate the FIC index,FIC=MICa drug combination/MICa drug use+MICb drugcombination/MICb drug use,standard of determining results:FIC index≤0.5,synergisticeffect;>0.5～1,additive effect;＞1～2,independent action;＞2,antagonism effect.Tubedouble dilution method is used to examine the best proportion of borneol and9kinds ofantibacterial agents for test strains,results showed that for Staphylococus aureus,therewere five kinds of combinations showed synergistic antibacterial effect,four kinds ofadditive effect, did not appear antagonist role;for drug-resistant Escherichiacoli(NO.13920),7kinds of combination showed synergistic antibacterial effect,1kind ofthem showedadditive effect,1for independent action, did not appear antagonist role;forpseudomonas aeruginosa (No.13930),6kinds of combination showed significantsynergistic effect,1for additive effect,2independent action,no antagonism effectappeared; for drug-resistant pneumonia klebsiella bacteria (No.13952),5kinds ofcombinations showed synergistic effect,2show additive effect,2independent action, didnot appear antagonism.The above results suggested that borneol can improve the effect ofantibacterial drugs of drug-resistant staphylococcus aureus, Escherichia coli,pseudomonas aeruginosa and klebsiella pneumonia bacterium infection when combineduse with part of antimicrobial agents.Both of the MIC of different ratio of gentamicin∕borneol, levofloxacin∕borneol,ceftazidime∕borneol used in conjunction weresignificantly lower than a single dose of antimicrobial agent,and4:1was the best ratio, itmeant that the antibacterial function of antimicrobial agents∕borneol was the best whenthe ratio was4:1.（4）To provide theoretical guidance for the study of the mechanism,clinical trialsand the development of new resistant preparations compound,the joint antibacterial effectof borneol and antibacterial in vivo and the best ratio of formula were studied.Kunmingmice (weight18-22g),male and female half,randomized based on theweight.By peritonealinjection for sepsis model with1MLD microbial which prepared by0.5mL highly active dry yeast,0.2mL different ratio of liquids was given by intragastric administrationimmediately,then observe and record apparent symptoms and survival rate of mice of eachdose after12,24,36,72h to7d.According to bliss method to calculate the drugs medianeffective dose(ED50) of bacteria infection in mice and95%confidence limits.Results oflevofloxacin and borneol in vivo antibacterial experiment showed that ED50oflevofloxacin single dose for staphylococcus aureus, escherichia coli, pseudomonasaeruginosa, pneumonia klebsiella bacteria infection sepsis model in mice were54.1,51.6,50.0,53.2㎎/㎏,respectively,while ED50of borneol were749.9,650.5,632.8,683.1㎎/kg.The ED50value of different ratio of levofloxacin and borneol were lower thanlevofloxacin single-dose on staphylococcus aureus, escherichia coli, pseudomonasaeruginosa, pneumonia klebsiella bacteria infection sepsis model in mice,which had ansignificant differences (P<0.05) after been analyzed by statistical processing.The result ofjoint antibacterial experiments of ceftazidime and borneol in vivo showed that ED50valuesof ceftazidime single dose for staphylococcus aureus, escherichia coli, pseudomonasaeruginosa, pneumonia klebsiella bacteria infection sepsis model in mice were944.7,975.0,882.1,1000.3㎎/㎏,respectively,and ED50values of different proportion ofceftazidime∕borneol were lower than ceftazidime single-dose,which showed a significantdifferences(P＜0.05) between them after been analyzed by statistical software.Through the study of this article,the joint antibacterial effect in vivo and in vitro ofborneol and antimicrobial drugs for four kinds of clinical drug-resistant strains werediscussed,and ED50values were determined,it will provide some theoretical guidance tomechanism research and clinical trials in the later research.